This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Ubiquitin-mediated proteolysis regulates a vast array of biological processes, including growth control and cancer, signal transduction, and the stress response. Ubiquitination of a target protein proceeds through a triple-enzyme cascade, entailing ubiquitin-activating (E1), conjugating (E2), and ligating (E3) activities. Despite intensive structural and functional analysis of this ubiquitination reaction, our current knowledge of how ubiquitin is transferred from E2 to particular lysine(s) of the bound substrate in E3 is extremely limited. The multisubunit SCF (Skp1-Cul1-Rbx1-F box protein) E2- and substrate-recruiting complexes comprise the largest superfamily of E3s. SCFFbx4 (the superscript denotes the F box protein) are responsible for regulated turnover of the cell-cycle activator cyclin D. High cyclin D levels are associated with high tumor grade, poor prognosis, and increased metastasis in many cancers. We are trying to get the crystal structures of SCFFbx4 alone and in complex with its substrate cyclin D and the E2 enzyme. The long-term objective of this work is to elucidate the structural and mechanistic basis for ubiquitin transfer by characterizing ubiquitination site selection, requirement for SCF dimerization, and E2-SCF interactions of the cyclin D ubiquitination reaction. Our studies will address both general aspects of the ubiquitination reaction by the SCF E3s and also specific questions about cyclin D degradations. The knowledge gained will provide a novel avenue for the development of therapeutics designed to manipulate cyclin D levels in cancer.
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